Ultrasonography (US) provides detailed visualization of the fetus in early pregnancy in cows, thus allowing for fetal sex determination. The objective of this prospective observational study was to determine the feasibility and accuracy of a single US examination to diagnose fetal sex in dairy cattle under routine reproductive management conditions. For this purpose, 953 Holstein cows at 7-16weeks of gestation were examined. Gender assignment was performed in 822 cows, while the genitalia could not be clearly visualized in 131 (13.7%) of the fetuses. After calving, it was verified that 99.3% of the diagnoses were accurate. Fetal sex was correctly determined by US in 99.5% of male fetuses and 98.8% of female fetuses. Fetal sex determination was less accurate when conducted before d 55 of gestation. Likewise, it was verified that fetal sex, cow age and ultrasonographic diagnosis section did not have a significant influence (P>0.05) on diagnostic accuracy. With respect to the plane used for diagnosis, the sagittal view was poorly used for early pregnancy diagnosis, whereas the longitudinal and cross-sectional planes were used most frequently. These results demonstrate that US can be routinely applied under farm conditions to accurately determine the fetal sex in cattle between days 51 and 111 of gestation without apparent influence of cow age, US scanning plane or fetal sex. Conversely, days of gestation affected the accuracy and feasibility of US gender determination, showing poorer results when the diagnosis was made before day 55 of gestation.